Journal of Chemical Ecology, VoL 9, No. 3, 1983
C H E M O T A X I S OF LARVAL SOYBEAN CYST N E M A T O D E , Heterodera glycines RACE 3, TO ROOT LEACHATES A N D IONS
MICHAEL
K. P A P A D E M E T R I O U
a n d L E O N W. B O N E 1
Department of Physiology, Southern Illinois University Carbondale , Illinois 62901
(Received June 3, 1982; revised August 10, 1982) Abstract--Second-stage larvae of the soybean cyst nematode Heterodera glycines Race 3 were attracted in an in vitro bioassay to pooled leachates of soybean roots that were expressed as root gram-hours (1 g of root/br/vol). Their responses were dosage dependent with maximal attraction to a 5 root g-hr/ml source. Optimal bioassay conditions used 24 hr for gradient formation and 1.5 hr for larval movement. Individual plants produced leachate with little variation in biological activity. Production was constant for five days of preparation. The attractive activity of root leachate declined with storage at 4~ C and heating over 30~ C, but loss of activity was reduced by freezing. Extraction and Sep-Pak fractionation indicated that the attractions(s) was water-soluble. Larvae were attracted also to several ionic solutions. Key Words--Soybean cyst nematode, nematode chemotaxis, larval attraction, Nematoda, host location.
INTRODUCTION A n u m b e r o f p l a n t s t i m u l i are p r o p o s e d to m e d i a t e t h e l a r v a l b e h a v i o r o f n e m a t o d e s f o r h o s t l o c a t i o n . C r o l l (1970) a n d G r e e n ( 1971) h a v e r e v i e w e d t h e r o l e o f v a r i o u s p h y s i c a l a n d c h e m i c a l f a c t o r s . S e v e r a l species o f H e t e r o d e r a l a r v a e h a v e b e e n s t u d i e d to d e t e r m i n e t h e c h e m i c a l basis o f t h e i r o r i e n t a t i o n to h o s t plants. H. s c h a c h t i i are a t t r a c t e d to c a r b o n d i o x i d e as a gas o r c a r b o x y l i o n , oxygen, potassium permangante, and reducing agents. Host plants and 1Present address : Regional Parasite Research Laboratory, USDA, P.O. Box 952, Auburn, Alabama 36380. 387 0098-033 l ] 83/0300-0387503,00] 0 9 1983 Plenum Publishing Corporation
388
PAPADEMETRIOU AND BONE
microorganisms were attractive while nonhost plants elicited only weak responses by larvae. H. avenae and H. rostochiensis respond also to exudates of host plants. The plant compounds are largely unknown, but aqueous components are implicated. Additionally, host attraction may occur over a 40-cm distance (Luc, 1961). Orientation to ionic gradients has been little studied. H. rostochiensis larvae were attracted to a m m o n i u m nitrate (Rode, 1965); however, electrical potentials are implicated for several other species of nematode larvae (Croll, 1970). Additionally many ions stimulate hatching of Heterodera eggs (Clarke and Shepherd, 1966). Chemotaxis of larvae of the soybean cyst nematode, Heterodera glyeines, has not been examined despite its economic importance. Thus, preliminary investigations of the larval response to exudates of host plants and ions were conducted to further our understanding of chemically mediated events in the host-parasite association.
M E T H O D S AND MATERIALS
Heterodera glycines Race 3 were maintained on soybean plants as previously reported (Rende et al., 1982). Cysts were opened manually and second-stage larvae were collected for bioassay within 48 hr after hatching from eggs. Bioassay was performed in l0 • 1.5-cm plastic Petri dishes which were coated with a thin file of 1.5% agar in reagent-grade (18 ml~) water from a Milli-Q purification system. Filter paper circles (Whatman No. 1) with a 7-mm diameter were placed in the center of the dish. Two circles were stacked in each dish and 1.5 #1 of the test solution was placed on the paper. The bioassay was modified from Schmidt and All (1978). After a 24-hr diffusion period, larvae were positioned 8 m m from the paper disk. Three larvae, placed equidistant from one another, were used on each plate. Attraction was determined as the percentage of larvae that reached the filter paper in the response period, according to analysis of their tracks. Initially, larva1 responses to root leachate were examined. Concentrations were prepared according to Tefft et al. (1982) and expressed as root gram-hours/unit volume. Six to eight plants were used for preparation of a standard solution in distilled water, based on estimated root mass. Various dosages from 0.5 to 15 root g-hr were tested with at least 30 replicates per dose. Preliminary trials examined larval responses at 10-rain intervals from 10 to 120 rain after 24 hr of diffusion to determine the time of maximal responsiveness. Subsequent experiments employed 1.5 hr for response. Distilled water was tested as a control. The influence of diffusion period on the larval responses to roots was
CHEMOTAXIS OF SOYBEAN NEMATODE
389
investigated. Leachate was prepared at 3 root g-hr for bioassay after 2, 3, 6, 8, 24, and 48 hr of chemical diffusion. Thirty replicates were done for each period of diffusion. Other experiments studied various factors that potentially influenced the production, stability, and characterization of the chemoattractant from soybean roots. Stability of the chemotactic activity of the leachate was examined by storage of solutions at 4 ~ C for 18 days. Bioassay was done at a dosage of 7.3 root g-hr to evaluate remaining attractancy for larvae after 1, 4, 6, 11, and 18 days. Leachate was held also under vacuum for 15 min to assay any volatility of the attractants by similar bioassay procedures at a dosage of 15 root g-hr. Thermal lability of the attractant from leachate was determined by heating at 30, 35, 40, 50, and 60 ~ C for 15 min. Bioassay at a dosage of 6 root g-hr was done after cooling for comparison to the standard dose-response line at ambient (20-22~ temperature. Frozen leachate was tested also by bioassay at dosages of 2 and 5.6 root g-hr after 14 days of storage to further assess stability. Preliminary fractionation of root leachate was initiated by extraction with a threefold volume of chloroform-methanol (2: 1). After nitrogen evaporation of the organic layer, the residue was dissolved in distilled water. This reconstituted material and the aqueous layer were tested for biological activity by dosage assay. Reverse-phase Sep-Paks (Waters Associates) were also employed for separation of leachate components. Five milliliters of solution of 3.8 root g-hr/ml were eluted through the C-18 cartridge. Retained compounds were then eluted with distilled water or 25, 50, 75, and 100% methanol. After solvent evaporation and reconstitution in distilled water, these fractions were assayed at 3.8 root g-hr to determine larval responses with 30 replicates per fraction. The attractancy of individual plants was determined also. After preparation of leachate, the resultant solutions were adjusted to 2.25 or 5.6 root g-hr concentrations with distilled water. Larval responses were examined by bioassay of 30 replicates per plant. Six plants were tested at each of the above doses for comparison to the standard dose-response line from pooled leachate. The release or production of the attractant in root leachate was studied by incubation of root mass for 1, 2, 3, 4, or 5 days. Each leachate was then adjusted to a 3.9 root g-hr dosage for bioassay of 30 replicates of the larval response. Additionally, the orientation of larval H. glycines toionic solutions was studied. Tested solutes included sodium sulfate, sodium chloride, magnesium sulfate, magnesium chloride, zinc sulfate, zinc chloride, ferric sulfate, ferric chloride, calcium sulfate, and calcium chloride. A range of at least five dosages from approximately 2 to 250 mM were done for each solution with 30
390
PAPADEMETRIOU AND BONE
replicates per dose. Bioassay procedures were similar to those for root leachate. Distilled, reagent-grade (18 mfl) water was used as a control. Data were analyzed by linear regression and analysis of variance. The 0.05 probability was considered significant. RESULTS Responses of larval H. glycines to dosages of soybean roots are shown in Figure 1 for a 24-hr diffusion period and a 1.5-hr period for larval orientation. Larvae were significantly attracted to the root solution, based on dosage (r = 0.80). Maximal response (63%) was caused by a 3 root g-hr/ml dosage in comparison to the 24% responses to the distilled water or filter paper controls. Dosages over 0.5 g-hr were significantly different from zero (SEM = 5.57) Assay of dosages to 15 root g-hr elicited responses similar to 3-5 g-hr concentrations.
70"
60-
50Z
O 40-
30-
20-
10-
--"r-C/
o
,
o.s
i
ROOT LEACHATE (gram-hours)
FIG. 1. Responses of H. glycines larvae to dosages of soybean root leachate in gram-hours after 24-hr diffusion and 1.5-hr response periods (SEM = 5.57).
CHEMOTAXIS OF SOYBEAN NEMATODE
391
70-
3gram-hours
60500Z 40m
....io -ur-s--ogram o/
,----4-----*-
....
20-
I0- o~/~''" 10
210 310 410 510 610 710 810 90 100 1~0 120 RESPONSE TIME(MIN)
FTG. 2. Timed responses of H. glycines larvae to selected dosages of soybean root leachate at 0.5 (e) g-hr (SEM = 2.72, r = 0.96) and 3 (0) g-hr (SEM = 5.77, r = 0.98). Figure 2 gives the effect of root dosage on the larval response time for selected dosages. Generally, maximal responses were obtained after a 90- or 100-min period. The percent of larval attraction increased linearly from 10 to 90 min, but showed little change with additional time. Lower dosages were intermediate to those in Figure 2. Diffusion time for the root solution influenced larval responsiveness (Figure 3). Insignificant responses were found with 8 hr or less for chemical diffusion when compared to controls (SEM = 3.7). Maximal responsiveness by larvae occurred in a 24-hr gradient, while a 48-hr period reduced larval attraction slightly presumably due to gradient equilibrium. The attractiveness of the root leachate for larvae declined as storage time increased (Figure 4). Insignificant responses were obtained with 10-day or older leachate (SEM = 6.2) when compared to the controls. Larval responses to the original and evacuated leachates were similar at the examined dose. Thus volatility of the root attractant(s) appears negligible. Increased temperature reduced linearly (r = 0.98) the attractiveness of the root leachate according to bioassay (Figure 5). No remaining activity was found after heating at 60~ for 15 min. Moderate temperature (30~
392
PAPADEMETRIOU AND BONE
40-
30-
20-
10-
i
!
468
I
2',
.'8
DIFFUSION TIME (HR)
FIG. 3. Larva] responses of H. glycines to a 3 root g-hr dosage after the indicated diffusion periods and a 1.5-hr response period ( S E M = 3.7).
50@
40-
z
O O_
30-
IX
20-
10-
|
I
!
!
I
1
3
5
10
18
LEACHATE STORAGE (DAYS) Fro. 4. Larval responses of H. glycines to soybean r o o t leachate after storage at 4 ~ C for the indicated periods ( S E M = 6.2).
393
C H E M O T A X I S OF SOYBEAN N E M A T O D E
40-
30-
2o-
10-
3'0
3'5
4'o
'
s'o
'
TEMPERATURE (*C)
FIG. 5. Larval responses of H. glycines to soybean root leachate after heating for 15 min at the indicated temperatures (SEM = 3.86). however, caused no appreciable change when compared to previous results from bioassay at ambient temperatures. Bioassay of frozen leachate after 14 days of storage revealed no appreciable change in attractiveness according to the larval responses. Thus, a 5.6 root g-hr dose elicited an initial response of 38% and an elevated 52% response after freezing. Therefore, freezing may partially reduce the loss of leachate attractancy that occurs at 4~ C or elevated temperatures. Extraction of the root leachate with organic solvents revealed that the biological activity for larval attraction remained in the aqueous fraction. This fraction caused a dosage-dependent attraction (r -- 0.81) of the larvae with a maximal 39% response to a 10 root g-hr dosage. This decreased response, however, suggests some activity was lost. In contrast, the reconstituted organic fraction did not differ from the control responses (24%) until a 36 root g-hr dose was exceeded. A 47% response occurred at 45 root g-hr of the organic fraction. This represents only 4% of the expected activity and therefore may have resulted from inadequate extraction or manipulative error. Similar results were obtained by Sep-Pak fraction. Elutions showed that the aqueous fraction contained most of the biological activity of the original leachate and caused a 67% response by larvae. The methanol eluants (mean = 25.5%) did not differ significantly from the control responses to distilled water. Thus, the attractant is probably water-soluble, based on extraction and separation data. Little variation was found in the larvae's response to leachate that was prepared from individual plants. The percent response to a 2.25 g-hr dose
394
PAPADEMETRIOU AND BONE
ranged from 29 to 34% for five tested plants while the sixth plant caused only a 17% attraction. The mean response (29.3%, SEM = 2.32) agreed with that from pooled samples. At 5.6 g-hr the larval responses to five individual hosts were 36-43% except for a single plant that elicited a 17% response. The mean (34.5%, SEM = 3.35) was similar to that from pooled leachate (Figure 1). No significant decrease in the production of attractant in leachate solution was observed during 1-5 days of preparation (r = 0.59) (Figure 6). A tendency to decreased activity, however, was suggested at longer periods. Other studies of this investigation employed a 1- or 2-day preparation of leachate. Little difference occurred at these times. Based on the responses to distilled water versus reagent-grade water as controls (24 and 13%, respectively), larval orientaiton to ions was examined also. Larvae showed no significant response to the sulfates or chlorides of sodium and iron. Dosage-dependent responses by larvae, however, were found for zinc sulfate, zinc chloride, calcium sulfate and magnesium chloride (r = 0.96, 0.79, 0.79, and 0.83, respectively). The threshold for responsiveness was lowest for calcium sulfate (1.5 mM) and highest for zinc chloride (36 mM). Increased responsiveness did not occur beyond a 60- to 70-mM concentration of the ionic solution. Thus, the larvae of H. glyeines are
60-
50-
40IJJ r Z
o
uJ r162
30-
20-
10-
i
I
I
I
2
3
,
ROOT INCUBATION (DAYS)
FIG. 6. Larval responses of H. glycines to soybean root leachate that was prepared by plant incubation for the indicated periods (SEM = 3.29).
CHEMOTAXISOF SOYBEANNEMATODE
395
attracted to various ions which may account for the twofold difference in the control responses of this study.
DISCUSSION
Location of a host plant by larval Heterodera glycines is partially mediated by chemicals from roots. The c o m p o u n d is apparently watersoluble, nonvolatile, and heat-labile. These features are probably consistent with its chemical function in a subterranean environment. The loss of activity with increased temperature may be minor in reduced soil temperatures. Also, we suspect that the loss of activity with storage and incubation time of the plant pose no problem in the natural ecology of parasitism; these features are probably more important during experimental treatments. Ionic gradients may be somewhat involved, but the lability of root leachates reinforces the presence of additional biochemical components. The larvae's attraction to certain ions is particularly interesting since many of these substances serve as hatching stimuli in various species of cyst nematodes. The response of the soybean cyst nematode to zinc is intriguing due to the role of this ion as one of the few known stimuli that induce hatching. If zinc is involved to some degree in both egg hatching and host location, its role as a biological messenger becomes quite plausible. However, further studies that are directed toward isolation of other active material are needed for more comprehensive knowledge. The consistent production of the compound(s) by individual plants for a period of time may contribute to plant susceptibility or levels of nematode infection. Examination of the orientation of larvae to root leachates of nonhost plants or resistant cultivars of soybeans may offer additional insight into host-parasite interaction with a resultant agricultural impact. Acknowledgments--Research was supported by funds from the Office of Research Development and Administration, Southern Illinois University-Carbondale.
REFERENCES
CLARKE,A.J., and SHEPHERD,A.M. 1966. Inorganic ions and the hatching of Heterodera spp. Ann. Appl. Biol. 58:497-508. CROLL, N.A. 1970. The Behavior of Nematodes. St. Martins, New York, 117 pp. GREEN, C.D. 1971. Mating and host finding behavior of plant nematodes, pp. 247-266, in B.M. Zuckerman, W.F. Mai, and R.A. Rohde (eds.). Plant Parasitic Nematodes. Academic Press, New York. LtJc, M. 1961. Note preliminaire sur le deplaeement de Hemicycliophora paradoxa Luc (Nematoda, Criconematidae) dans le sol. Nematologica 6:95-106.
396
PAPADEMETRIOU AND BONE
RENDE, J.F., TEFFT, P.M., and BONE, L.W. 1981. Pheromone attraction in the soybean cyst nematode Heterodera glycines Race 3. J. Chem. Ecol. 8:981-991.
RODE, n . 1965. Uber einige Methoden zur Verhaltensforschung bei Nematoden. Pedobiologica 5:1-16. SCHMIDT, J., and ALL, J.N. 1978. Chemical attraction of Neoaplectana carpocapsae (Nematoda: Steinernematidae) to insect larvae. Environ. Entomol. 7:605-607. TEFFT, P.M.~ RENDE, J.F., and BONE, L.W. 1982. Factors influencing egg hatching of the soybean cyst nematode Heterodera glycines Race 3. Proc. Helminithol Soc. Wash. 49:258-265.
C H E M O T A X I S OF LARVAL SOYBEAN CYST N E M A T O D E , Heterodera glycines RACE 3, TO ROOT LEACHATES A N D IONS
MICHAEL
K. P A P A D E M E T R I O U
a n d L E O N W. B O N E 1
Department of Physiology, Southern Illinois University Carbondale , Illinois 62901
(Received June 3, 1982; revised August 10, 1982) Abstract--Second-stage larvae of the soybean cyst nematode Heterodera glycines Race 3 were attracted in an in vitro bioassay to pooled leachates of soybean roots that were expressed as root gram-hours (1 g of root/br/vol). Their responses were dosage dependent with maximal attraction to a 5 root g-hr/ml source. Optimal bioassay conditions used 24 hr for gradient formation and 1.5 hr for larval movement. Individual plants produced leachate with little variation in biological activity. Production was constant for five days of preparation. The attractive activity of root leachate declined with storage at 4~ C and heating over 30~ C, but loss of activity was reduced by freezing. Extraction and Sep-Pak fractionation indicated that the attractions(s) was water-soluble. Larvae were attracted also to several ionic solutions. Key Words--Soybean cyst nematode, nematode chemotaxis, larval attraction, Nematoda, host location.
INTRODUCTION A n u m b e r o f p l a n t s t i m u l i are p r o p o s e d to m e d i a t e t h e l a r v a l b e h a v i o r o f n e m a t o d e s f o r h o s t l o c a t i o n . C r o l l (1970) a n d G r e e n ( 1971) h a v e r e v i e w e d t h e r o l e o f v a r i o u s p h y s i c a l a n d c h e m i c a l f a c t o r s . S e v e r a l species o f H e t e r o d e r a l a r v a e h a v e b e e n s t u d i e d to d e t e r m i n e t h e c h e m i c a l basis o f t h e i r o r i e n t a t i o n to h o s t plants. H. s c h a c h t i i are a t t r a c t e d to c a r b o n d i o x i d e as a gas o r c a r b o x y l i o n , oxygen, potassium permangante, and reducing agents. Host plants and 1Present address : Regional Parasite Research Laboratory, USDA, P.O. Box 952, Auburn, Alabama 36380. 387 0098-033 l ] 83/0300-0387503,00] 0 9 1983 Plenum Publishing Corporation
388
PAPADEMETRIOU AND BONE
microorganisms were attractive while nonhost plants elicited only weak responses by larvae. H. avenae and H. rostochiensis respond also to exudates of host plants. The plant compounds are largely unknown, but aqueous components are implicated. Additionally, host attraction may occur over a 40-cm distance (Luc, 1961). Orientation to ionic gradients has been little studied. H. rostochiensis larvae were attracted to a m m o n i u m nitrate (Rode, 1965); however, electrical potentials are implicated for several other species of nematode larvae (Croll, 1970). Additionally many ions stimulate hatching of Heterodera eggs (Clarke and Shepherd, 1966). Chemotaxis of larvae of the soybean cyst nematode, Heterodera glyeines, has not been examined despite its economic importance. Thus, preliminary investigations of the larval response to exudates of host plants and ions were conducted to further our understanding of chemically mediated events in the host-parasite association.
M E T H O D S AND MATERIALS
Heterodera glycines Race 3 were maintained on soybean plants as previously reported (Rende et al., 1982). Cysts were opened manually and second-stage larvae were collected for bioassay within 48 hr after hatching from eggs. Bioassay was performed in l0 • 1.5-cm plastic Petri dishes which were coated with a thin file of 1.5% agar in reagent-grade (18 ml~) water from a Milli-Q purification system. Filter paper circles (Whatman No. 1) with a 7-mm diameter were placed in the center of the dish. Two circles were stacked in each dish and 1.5 #1 of the test solution was placed on the paper. The bioassay was modified from Schmidt and All (1978). After a 24-hr diffusion period, larvae were positioned 8 m m from the paper disk. Three larvae, placed equidistant from one another, were used on each plate. Attraction was determined as the percentage of larvae that reached the filter paper in the response period, according to analysis of their tracks. Initially, larva1 responses to root leachate were examined. Concentrations were prepared according to Tefft et al. (1982) and expressed as root gram-hours/unit volume. Six to eight plants were used for preparation of a standard solution in distilled water, based on estimated root mass. Various dosages from 0.5 to 15 root g-hr were tested with at least 30 replicates per dose. Preliminary trials examined larval responses at 10-rain intervals from 10 to 120 rain after 24 hr of diffusion to determine the time of maximal responsiveness. Subsequent experiments employed 1.5 hr for response. Distilled water was tested as a control. The influence of diffusion period on the larval responses to roots was
CHEMOTAXIS OF SOYBEAN NEMATODE
389
investigated. Leachate was prepared at 3 root g-hr for bioassay after 2, 3, 6, 8, 24, and 48 hr of chemical diffusion. Thirty replicates were done for each period of diffusion. Other experiments studied various factors that potentially influenced the production, stability, and characterization of the chemoattractant from soybean roots. Stability of the chemotactic activity of the leachate was examined by storage of solutions at 4 ~ C for 18 days. Bioassay was done at a dosage of 7.3 root g-hr to evaluate remaining attractancy for larvae after 1, 4, 6, 11, and 18 days. Leachate was held also under vacuum for 15 min to assay any volatility of the attractants by similar bioassay procedures at a dosage of 15 root g-hr. Thermal lability of the attractant from leachate was determined by heating at 30, 35, 40, 50, and 60 ~ C for 15 min. Bioassay at a dosage of 6 root g-hr was done after cooling for comparison to the standard dose-response line at ambient (20-22~ temperature. Frozen leachate was tested also by bioassay at dosages of 2 and 5.6 root g-hr after 14 days of storage to further assess stability. Preliminary fractionation of root leachate was initiated by extraction with a threefold volume of chloroform-methanol (2: 1). After nitrogen evaporation of the organic layer, the residue was dissolved in distilled water. This reconstituted material and the aqueous layer were tested for biological activity by dosage assay. Reverse-phase Sep-Paks (Waters Associates) were also employed for separation of leachate components. Five milliliters of solution of 3.8 root g-hr/ml were eluted through the C-18 cartridge. Retained compounds were then eluted with distilled water or 25, 50, 75, and 100% methanol. After solvent evaporation and reconstitution in distilled water, these fractions were assayed at 3.8 root g-hr to determine larval responses with 30 replicates per fraction. The attractancy of individual plants was determined also. After preparation of leachate, the resultant solutions were adjusted to 2.25 or 5.6 root g-hr concentrations with distilled water. Larval responses were examined by bioassay of 30 replicates per plant. Six plants were tested at each of the above doses for comparison to the standard dose-response line from pooled leachate. The release or production of the attractant in root leachate was studied by incubation of root mass for 1, 2, 3, 4, or 5 days. Each leachate was then adjusted to a 3.9 root g-hr dosage for bioassay of 30 replicates of the larval response. Additionally, the orientation of larval H. glycines toionic solutions was studied. Tested solutes included sodium sulfate, sodium chloride, magnesium sulfate, magnesium chloride, zinc sulfate, zinc chloride, ferric sulfate, ferric chloride, calcium sulfate, and calcium chloride. A range of at least five dosages from approximately 2 to 250 mM were done for each solution with 30
390
PAPADEMETRIOU AND BONE
replicates per dose. Bioassay procedures were similar to those for root leachate. Distilled, reagent-grade (18 mfl) water was used as a control. Data were analyzed by linear regression and analysis of variance. The 0.05 probability was considered significant. RESULTS Responses of larval H. glycines to dosages of soybean roots are shown in Figure 1 for a 24-hr diffusion period and a 1.5-hr period for larval orientation. Larvae were significantly attracted to the root solution, based on dosage (r = 0.80). Maximal response (63%) was caused by a 3 root g-hr/ml dosage in comparison to the 24% responses to the distilled water or filter paper controls. Dosages over 0.5 g-hr were significantly different from zero (SEM = 5.57) Assay of dosages to 15 root g-hr elicited responses similar to 3-5 g-hr concentrations.
70"
60-
50Z
O 40-
30-
20-
10-
--"r-C/
o
,
o.s
i
ROOT LEACHATE (gram-hours)
FIG. 1. Responses of H. glycines larvae to dosages of soybean root leachate in gram-hours after 24-hr diffusion and 1.5-hr response periods (SEM = 5.57).
CHEMOTAXIS OF SOYBEAN NEMATODE
391
70-
3gram-hours
60500Z 40m
....io -ur-s--ogram o/
,----4-----*-
....
20-
I0- o~/~''" 10
210 310 410 510 610 710 810 90 100 1~0 120 RESPONSE TIME(MIN)
FTG. 2. Timed responses of H. glycines larvae to selected dosages of soybean root leachate at 0.5 (e) g-hr (SEM = 2.72, r = 0.96) and 3 (0) g-hr (SEM = 5.77, r = 0.98). Figure 2 gives the effect of root dosage on the larval response time for selected dosages. Generally, maximal responses were obtained after a 90- or 100-min period. The percent of larval attraction increased linearly from 10 to 90 min, but showed little change with additional time. Lower dosages were intermediate to those in Figure 2. Diffusion time for the root solution influenced larval responsiveness (Figure 3). Insignificant responses were found with 8 hr or less for chemical diffusion when compared to controls (SEM = 3.7). Maximal responsiveness by larvae occurred in a 24-hr gradient, while a 48-hr period reduced larval attraction slightly presumably due to gradient equilibrium. The attractiveness of the root leachate for larvae declined as storage time increased (Figure 4). Insignificant responses were obtained with 10-day or older leachate (SEM = 6.2) when compared to the controls. Larval responses to the original and evacuated leachates were similar at the examined dose. Thus volatility of the root attractant(s) appears negligible. Increased temperature reduced linearly (r = 0.98) the attractiveness of the root leachate according to bioassay (Figure 5). No remaining activity was found after heating at 60~ for 15 min. Moderate temperature (30~
392
PAPADEMETRIOU AND BONE
40-
30-
20-
10-
i
!
468
I
2',
.'8
DIFFUSION TIME (HR)
FIG. 3. Larva] responses of H. glycines to a 3 root g-hr dosage after the indicated diffusion periods and a 1.5-hr response period ( S E M = 3.7).
50@
40-
z
O O_
30-
IX
20-
10-
|
I
!
!
I
1
3
5
10
18
LEACHATE STORAGE (DAYS) Fro. 4. Larval responses of H. glycines to soybean r o o t leachate after storage at 4 ~ C for the indicated periods ( S E M = 6.2).
393
C H E M O T A X I S OF SOYBEAN N E M A T O D E
40-
30-
2o-
10-
3'0
3'5
4'o
'
s'o
'
TEMPERATURE (*C)
FIG. 5. Larval responses of H. glycines to soybean root leachate after heating for 15 min at the indicated temperatures (SEM = 3.86). however, caused no appreciable change when compared to previous results from bioassay at ambient temperatures. Bioassay of frozen leachate after 14 days of storage revealed no appreciable change in attractiveness according to the larval responses. Thus, a 5.6 root g-hr dose elicited an initial response of 38% and an elevated 52% response after freezing. Therefore, freezing may partially reduce the loss of leachate attractancy that occurs at 4~ C or elevated temperatures. Extraction of the root leachate with organic solvents revealed that the biological activity for larval attraction remained in the aqueous fraction. This fraction caused a dosage-dependent attraction (r -- 0.81) of the larvae with a maximal 39% response to a 10 root g-hr dosage. This decreased response, however, suggests some activity was lost. In contrast, the reconstituted organic fraction did not differ from the control responses (24%) until a 36 root g-hr dose was exceeded. A 47% response occurred at 45 root g-hr of the organic fraction. This represents only 4% of the expected activity and therefore may have resulted from inadequate extraction or manipulative error. Similar results were obtained by Sep-Pak fraction. Elutions showed that the aqueous fraction contained most of the biological activity of the original leachate and caused a 67% response by larvae. The methanol eluants (mean = 25.5%) did not differ significantly from the control responses to distilled water. Thus, the attractant is probably water-soluble, based on extraction and separation data. Little variation was found in the larvae's response to leachate that was prepared from individual plants. The percent response to a 2.25 g-hr dose
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ranged from 29 to 34% for five tested plants while the sixth plant caused only a 17% attraction. The mean response (29.3%, SEM = 2.32) agreed with that from pooled samples. At 5.6 g-hr the larval responses to five individual hosts were 36-43% except for a single plant that elicited a 17% response. The mean (34.5%, SEM = 3.35) was similar to that from pooled leachate (Figure 1). No significant decrease in the production of attractant in leachate solution was observed during 1-5 days of preparation (r = 0.59) (Figure 6). A tendency to decreased activity, however, was suggested at longer periods. Other studies of this investigation employed a 1- or 2-day preparation of leachate. Little difference occurred at these times. Based on the responses to distilled water versus reagent-grade water as controls (24 and 13%, respectively), larval orientaiton to ions was examined also. Larvae showed no significant response to the sulfates or chlorides of sodium and iron. Dosage-dependent responses by larvae, however, were found for zinc sulfate, zinc chloride, calcium sulfate and magnesium chloride (r = 0.96, 0.79, 0.79, and 0.83, respectively). The threshold for responsiveness was lowest for calcium sulfate (1.5 mM) and highest for zinc chloride (36 mM). Increased responsiveness did not occur beyond a 60- to 70-mM concentration of the ionic solution. Thus, the larvae of H. glyeines are
60-
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30-
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ROOT INCUBATION (DAYS)
FIG. 6. Larval responses of H. glycines to soybean root leachate that was prepared by plant incubation for the indicated periods (SEM = 3.29).
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attracted to various ions which may account for the twofold difference in the control responses of this study.
DISCUSSION
Location of a host plant by larval Heterodera glycines is partially mediated by chemicals from roots. The c o m p o u n d is apparently watersoluble, nonvolatile, and heat-labile. These features are probably consistent with its chemical function in a subterranean environment. The loss of activity with increased temperature may be minor in reduced soil temperatures. Also, we suspect that the loss of activity with storage and incubation time of the plant pose no problem in the natural ecology of parasitism; these features are probably more important during experimental treatments. Ionic gradients may be somewhat involved, but the lability of root leachates reinforces the presence of additional biochemical components. The larvae's attraction to certain ions is particularly interesting since many of these substances serve as hatching stimuli in various species of cyst nematodes. The response of the soybean cyst nematode to zinc is intriguing due to the role of this ion as one of the few known stimuli that induce hatching. If zinc is involved to some degree in both egg hatching and host location, its role as a biological messenger becomes quite plausible. However, further studies that are directed toward isolation of other active material are needed for more comprehensive knowledge. The consistent production of the compound(s) by individual plants for a period of time may contribute to plant susceptibility or levels of nematode infection. Examination of the orientation of larvae to root leachates of nonhost plants or resistant cultivars of soybeans may offer additional insight into host-parasite interaction with a resultant agricultural impact. Acknowledgments--Research was supported by funds from the Office of Research Development and Administration, Southern Illinois University-Carbondale.
REFERENCES
CLARKE,A.J., and SHEPHERD,A.M. 1966. Inorganic ions and the hatching of Heterodera spp. Ann. Appl. Biol. 58:497-508. CROLL, N.A. 1970. The Behavior of Nematodes. St. Martins, New York, 117 pp. GREEN, C.D. 1971. Mating and host finding behavior of plant nematodes, pp. 247-266, in B.M. Zuckerman, W.F. Mai, and R.A. Rohde (eds.). Plant Parasitic Nematodes. Academic Press, New York. LtJc, M. 1961. Note preliminaire sur le deplaeement de Hemicycliophora paradoxa Luc (Nematoda, Criconematidae) dans le sol. Nematologica 6:95-106.
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RENDE, J.F., TEFFT, P.M., and BONE, L.W. 1981. Pheromone attraction in the soybean cyst nematode Heterodera glycines Race 3. J. Chem. Ecol. 8:981-991.
RODE, n . 1965. Uber einige Methoden zur Verhaltensforschung bei Nematoden. Pedobiologica 5:1-16. SCHMIDT, J., and ALL, J.N. 1978. Chemical attraction of Neoaplectana carpocapsae (Nematoda: Steinernematidae) to insect larvae. Environ. Entomol. 7:605-607. TEFFT, P.M.~ RENDE, J.F., and BONE, L.W. 1982. Factors influencing egg hatching of the soybean cyst nematode Heterodera glycines Race 3. Proc. Helminithol Soc. Wash. 49:258-265.
